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The experimental was carried out on two sugarcane varieties, namely CoN -05071 and CoC-671 at the Sugarcane Tissue Culture Laboratory of Main Sugarcane Research Station, Navsari Agricultural University, Navsari during 2016-017. The investigation deals with protoplast fusion such as agglutinated protoplasts, and heterkaryon formation And Molecular character. In this experiment, PEG 6000(19%) has been found to be better than PEG 4000, CaCl2.2H2O(35μm) and 10.5pH was found to be better for total agglutination and heterokaryon formation in sugarcane cultivars. Somatic hybridization in a polyploid and vegetatively propagated crop such as sugarcane has a good potential for producing useful variation. Maximum polymorphism is observed among the genotypes and has accumulated maximum genetic changes in comparison.